Many biological functions, including antigenic functions, hormonal functions, enzymatic functions, and cell-regulatory functions are provided by proteins. Proteins consist of long chains of amino acids in a particular sequence. The above-mentioned functions are typically attributable to rather limited segments of the protein comprising short sequences of amino acids. The rest of the protein molecule often serves as a carrier for the functional segment or segments. The carrier segments protect the functional segments of the protein and present the functional segments to substrates in an orientation which promotes activity. In addition, certain properties of the functional segments of the protein are only able to take effect when the short sequences of amino-acids comprising the functional segments are connected to a longer protein chain. For example, immune response to a particular short amino-acid sequence generally requires that the short sequence be coupled to an extended molecule. In principle, the carrier segments of the protein could be replaced with a variety of other carrier segments without altering the properties of the functional protein segment. Such substitutions may effect certain distinct advantages, as will become clear, in the utilization of functional protein segments for commercial or medical purposes, such as the production of useful vaccines.
In particular, it will be seen that viral proteins are particularly suited to being exploited as carriers for small amino-acid sequences possessing useful functions. One particularly useful function of proteins, typically attributable to limited segments of a protein, is the ability to induce an immune response. When injected, inhaled, ingested, or otherwise placed into a live animal, a foreign protein, i.e., one not naturally present in the host animal, elicits an immune response. The immune response consists of many different concerted processes in the animal, including the production of antibodies, which attack the foreign protein and thereby protect the animal from infection by a carrier of the foreign protein. Importantly, an additional feature of the immune response is a form of biological memory such that a second exposure to the same foreign protein results in a quicker and much stronger immune response. This is the principle of vaccination which is an important part of modern medicine.
It has been found that effective immune responses are induced by small segments of proteins when they are attached to large carrier segments even if the carrier segments are not naturally of the same protein. Vaccinations with such proteins having a functional segment from one protein attached to unnatural carrier segments results not only in protection against further injection of the hybrid protein but also against the original protein from which the functional segment was obtained.
Typically, vaccines are produced in laboratories by preparing agents having substantially reduced pathogenicity with respect to disease-causing viruses that contain protein segments that induce an immune response. These agents are either strains of microorganisms which produce only mild diseases or else are chemically inactivated microorganisms. The vaccines are introduced into an animal to induce an immune response in the injected animal; however, there have been problems with such vaccines. Many infectious agents are difficult or impossible to grow under controlled conditions, and those which are grown and then inactivated present the possiblity of partial escape from the inactivation process which poses an appreciable risk to the vaccinated animal. With weakened strains of infectious microorganisms, the risk of natural mutation to more dangerous forms is inherent, similarly potentially endangering the vaccinated animal. Moreover, all the techniques involved in the production of such vaccines are time-consuming and expensive.
Accordingly, it is advantageous to use, as vaccines, immunogenic (immune-response-producing) protein segments obtained from infectious agents attached to unnatural carriers in place of the infectious agents themselves. In accordance with one aspect of the invention, viral proteins are particularly useful as carriers, and immunogenic protein segments are inserted into viral proteins in such a way that the viruses carry the segments so that they will be exposed to the immune system of a vaccinated animal without the immunogenic protein segments interfering with viral viability or reproduction. Several kinds of viruses can be used to carry immunogenic protein segments, each with distinct advantages. Among these are DNA-containing bacteriophages, nonpathogenic DNA-containing animal viruses and nonpathogenic enveloped RNA-containing influenza viruses.
DNA-containing bacteriophages, such as lambda phage, are viruses which infect bacteria. These viruses multiply to great numbers in bacteria, and they may be produced at small cost, are not pathogenic for animals or humans and can be introduced by ingestion, inhalation, or injection. Nonpathogenic animal viruses, such as the DNA-containing adenoviruses and the enveloped RNA-containing influenza viruses, replicate in human or animal cells, resulting in inapparent or inconsequential infections. These can thus be safely introduced by injection, ingestion or inhalation.
Proteins exposed on the surface of these viruses are preferred as foreign immunogenic protein segments. Surface proteins are capsid proteins in the case of non-enveloped viruses and trans-membrane proteins in the case of enveloped viruses.
When an immunogenic protein segment is incorporated in an exposed manner in a surface virus protein, the entire virus serves as an extended carrier. The virus carrier retains the ability to replicate while the incorporated foreign protein segment has the potential for inducing the specific immune response. The virus carrier also retains its biological functions, contributing to protein stability.
Other types of viruses may also be used to advantage as carriers in accordance with the invention. Furthermore, short protein segments with functions other than the capacity to stimulate immune responses may be incorporated as viral surface protein segments by the methods of the invention.
The joining of protein segments with specific functions to protein carriers may be accomplished by taking advantage of recent advances in understanding the genetic code, molecular biological processes and the technology of recombinant DNA genetics. The amino-acid sequences of cellular proteins, as well as most viral proteins, are determined by genes which are segments of deoxyribonucleic acids (DNA) sequenced according to the genetic code. The particular sequence of amino-acids is synthesized in accordance with the sequence of codons (triplets of nucleic acid subunits) in the DNA. Insertion of foreign DNA sequences into the DNA of a host organism, under certain appropriate conditions, results in the expression of the amino-acid sequence specified by the inserted, foreign DNA sequence.
Recombinant DNA technology allows such manipulations to be conveniently carried out. Sequences of DNA encoding a particular protein or protein segment may now be easily isolated and purified in large enough amounts to use biochemically. These sequences can then be cut in specific places, using enzymes known as restriction endonucleases, and spliced together with other purified fragments using DNA ligases. These recombinant molecules can then be put into living organisms, such as bacteria or higher cells.
It would be desirable to utilize the recombinant techniques which have been developed to incorporate protein segments having specific functions in surface viral proteins to provide useful agents for commercial and medical processes.
It is an object of the present invention to provide a method for attaching useful protein segments to virus carriers.
Another object of the invention is to provide viral carriers of immunogenic protein segments for inducing immune responses in animals. Specifically it is an object to produce new, safer vaccines. A further object is to provide improved vaccinations of mammals, including humans.